ABSTRACT
We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.
Subject(s)
DnaB Helicases , Genetics , Escherichia coli , Genetics , Metabolism , Factor VIII , Chemistry , Genetics , Metabolism , Inteins , Physiology , Peptide Fragments , Chemistry , Genetics , Metabolism , Protein Splicing , PhysiologyABSTRACT
PFW was isolated from fruit body of Pleurotus ferulae by using hot water Proteins were removed from PFW with Sevag method, after DEAE cellulose chromatography, PF 1 and PF 2 were isolated from PFW The weight was determined by using multiangle laser photometer combined with GPC; the results showed: PF 1 contains two fractions, the weights (Mw) are 7 875?10 5 and 3 245?10 4, the Mw of PF 2 is 3 187?10 6 The components and structure of polysaccharides were analyzed by IR and GC The polysaccharides PF 1 consists of rhamnose, glucose, galactose, mannose; PF 2 consists of rhamnose, xylose, glucose, galactose, mannose; PF 1 contains ? glucoside and mannoside, PF 2 contains ? glucoside It was found polysaccharides have antitumor activity on the implanted Sarcoma 180 in mice
ABSTRACT
Using GAD immunohistochemical PAP technique and microcomputer image analysis system, we have studied on the substantia nigra of rat epilep- tic model induced by Coriaria Lactone and discovered that in the early experi- mental group and 6 hour group, the intensity of GAD-reactivity of nerve fibers and terminals was weaker and the area density of GAD-positive nerve fibers and terminals was lower than those of control group (P